In the past two decades, the biological and medical fields have discovered the great advantages in the use of biosensors and biochips capable of characterizing and quantifying (bio)molecules. The fastest growing area in biosensors research involves affinity-based biosensors or immunosensors. These sensors are expected to revolutionize in areas like diagnostics, food processing, antiterrorism, environmental monitoring and public health where rapid detection combined to high sensitivity are important.
The development of strategies to immobilize groups of biomolecules to substrates has given rise to the field of biochips and has dramatically increased the rate and scope of discoveries in basic and applied science. A key challenge in biochip technology has been the development of reliable and reproducible chemistries for the immobilization of ligands or bioreceptors to a single substrate.
In order to be broadly useful for the preparation of a wide variety of biochips, the immobilization reaction should have several characteristics. First, the reaction should occur rapidly and therefore allow the use of low concentrations of reagents for immobilization. Second, the chemistry should require little, if any, post-synthetic modifications of ligands before immobilization to maximize the number of compounds that can be generated by solution or by solid-phase synthesis and minimize the cost of these reagents. Third, the immobilization process should occur selectively in the presence of common functional groups, including amines, thiols, carboxylic acids, and alcohols, to ensure that ligands are immobilized in a preferable oriented and homogeneous manner. Finally, the reaction should have well-behaved kinetics and be easily monitored with conventional spectroscopic methods to control the density of ligands on the chip.
Several chemical systems have been described and used for the immobilization of proteins to solid biochip surfaces. The protein coupling chemistry depends upon the underlying substrate of the biochip combined with the desired bioreceptor species one would like to couple to the biochip substrate. A number of methods have therefore been applied for the immobilization of receptor biomolecules, e.g. adsorption, covalent attachment to silanes or mixed layers of thiols, embedding in polymers and membranes. These different kinds of chemistries should be a compromise between the functional groups available on these chemistries and the functional groups available on the bioreceptor species, which one wants to immobilize on the biochip substrate. The groups on the bioreceptor species are important to achieve a random or orientated immobilization of the immobilized bioreceptors
It is known in the art of biosensors that molecules having the formula X—R—Ch-M adhered to a surface as part of a self assembled monolayer, where X is a functionality that adheres to the surface, R is a spacer moiety, M is a metal and Ch is a chelating agent for the metal ion M. These monolayers only have a limited surface accessibility for biological binding and oriented immobilization. Moreover, this type of monolayer can only be achieved via an extra cross-linking step.
A method for immobilizing proteins on mixed self-assembled monolayers of alkanethiols is also known in the art. This method needs an activation step comprising forming an N-hydroxysuccinimidyl (NHS) ester from the carboxylic acid groups of the self-assembled monolayer and then coupling this ester to a free amino group of the protein. In a first step, a self-assembling monolayer having free carboxylic acid groups is formed onto a gold surface. In a next step, the surface carboxylic acid groups are activated to form the NHS ester, followed by displacement of the NHS ester with an amino group of the protein to form an amido function. Since several steps have to be performed after deposition of the self-assembling monolayer onto the substrate, the yield reduces after each step, resulting in a low final yield of the immobilization degree.
Self-assembled monolayers with a CH═CH2 end group are also known in the art but no biomolecules or bioreceptors have been coupled to this functional group on a gold surface. In addition, such monolayers do not incorporate poly(ethylene oxide) groups which are desirable to avoid non-specific adsorption, a key issue in biosensing experiments.
Thiolate or disulfide self-assembled monolayers with aldehyde or epoxy end groups, which can be directly coupled to functional groups on biomolecules or bioreceptors are also known in the art. However these self-assembled monolayers do not incorporate poly(ethylene oxide) groups allowing a better sensitivity and specificity of the final biosensor interface.
Self-assembled monolayers with pre-activated groups, e.g. dithio-bis(succinimidylundecanoate), dithio-bis(succinimidylpropionate) and dithio-bis(succinimidylhexadecanoate) are already known in the art and can incorporate poly(ethylene oxide) groups. However the N-hydroxysuccinimidyl group is very sensitive to hydrolysis, which makes it difficult to store these activated samples before use.
Self-assembled monolayers incorporating poly(ethylene oxide) groups and a preactivated maleimidyl group are also known in the art. However a maleimide reacts preferentially with free thiol groups, which are not always readily available in proteins such as antibodies. In order to avoid this drawback, antibodies can be reduced to generate free thiol groups but this additional step is difficult to perform when forming biochips and often decreases the antibody affinity.
Health and environment related fields, faces various biochemical processes, which have to be evaluated rapidly at decreasing detection levels. Many biochemical analytical methods involve immobilization of a biological molecule on a surface. The increasing miniaturization and the demand for sensitivity require a covalent immobilization of biomolecules. Affinity biosensor transducers are defined as systems containing at least one biological element able to recognize an analyte. This element is called the biological recognition layer and consists of a probe molecule, covalently bound to a linking layer, which makes the connection with the transducer. The substrate can be a deposit of a metal film on any convenient support or any other solid surface able to selectively bind monolayers. Preferred metals include gold, silver, Ga—As alloys, palladium, platinum, copper, and the like. Silanes and alkyl phosphate monolayers can also be used on oxide material substrates like SiO2, Nb2O5, TiO2, ZrO2, Al2O3, and Ta2O5. A biosensor must respond to major qualities like stability, specificity, selectivity, and reproducibility. For all those reasons, only few affinity biosensors are commercially available. The major challenge is the realization of new specific and selective self-assembled monolayers and the receptors. An analyte must be detectable in an excess of other proteins. The most common receptors are antibodies and specific binding proteins which have a reversible specific binding affinity for an analyte. Chemical modifications of the surface moieties may create new surface functionalities, such as, for example, amine-terminated functional groups appropriate for particular diagnostic or therapeutic operations.